11beta-hydroxyl-16alpha-methyl steroids and 14alpha-hydroxyl-16alpha-methyl steroids



United States Patent Ofi ice 3,151,652 Patented Dec. 15, 1964 3,161,662 IIfl-HYDROXYL-lGa-METHYL STEROKDS AND 14a-HYDROXYL-16am 1ETHYL STEROIDS Klaus Kiesslich and Gerhard Pr'aspe, both of Berlin-Charlottenburg, Germany, assig'nors to Schenng A.G., Berlin, Germany 7 No Drawing. Filed May 8, 1961, Ser. No. 108,260 Claims priority, application Germany, May 13, 1960, Sch 27,885; Sept. 9, 1960, Sch 28,457; Feb. 3, 1961, Sch 29,168

5 Claims. (Cl. 260397.45)

l6a-methyl-4-pregnene-l 1p,17,2l-triol 3,20-dione (16amethyl-hydroeortisone) 6a-methyl-4-pregnene-l7a,2l diol 3,11,20 trione (i61- methylcortisone) lGrneIhyI-IH-preg'naZliene-I l 3,17 2 l-triol 3,20 dionc (ldd methyl' predni solone) dione' 16a=methyl fluorohydrocortisone).

It a primary object of the-present'invem' tion to provide a method of producing np=hydmxylated loan-methyl steroids in relatively liigh yield.

Itisanother objectof the present inilention to'pr'ovide' a method of producing new llB-hydroxylated Ion-methyl steroids.

It is yet anothe'robject of the present invention to pr'ovide for the production of new 14a-hydroxylated' 16ozmethyl steroids and to: provide for methods of producing these l4a-hydroxyl-l6ct-methyl steroids.

The ll B-hydrozrylated IGa-methyl steroids of the present invention have antiphlogistic, glucocorticoid and antiallergic properties but they do not exhibit mineralo-corticold or catabolic side efiects, which make these compounds extremely useful. In addition, these compounds can serve as intermediates in the snythesis of other valuable steroids, for example by acetylating any free hydroxyl group, by saturating any'double bond, etc.

The l4m-hydroxylated Mun-methyl steroids of the present invention also have useful properties as anti-phlogistic agents but not possessing typical corticoid activity. In addition, these l4a-hydroxylated l6rz-rnethyl steroids may serve as useful intermediates in the production of many useful steroids, for' example by saturating any unsaturated double bonds in known manner, by acetlylating or etherifying free hydroxyl groups, also in known manner, etc.

It is accordingly a further object of the present invention to provide new and useful steroid compounds.

Other objects and advantages of the present invention will be apparent from a further reading of the specification and of the appended claims.

With the above and otheh objects in view, the present invention mainly comprises with respect to the production of llB-hydroxylated mo -methyl stefoids-the subjecting of a Ida-methyl steroid of the prcgnane *ries to the action of a fungus strain which if used for llfihydroxylation of a l6a-methyl-iree steroid is known toform a side product with an a-oriented hydroxyl group in the D ring of the steroid, thereby forming the corresponding llp-hydroxylated -I6-m'ethyl steroid yield. p

The preferred for the production of the llfl-liydroxylaled lfia-methyl steroids of the present invention are the fungi oi the Curvularia strain, and particularly of the Curvula'ria lunata, as wellastheen zy mes flie reof.

In' addition to being applicable to the production of llp-hydroxyla ted-lm inethylsteroids thernthod of'the invention to the production of new ll'fl-liydroiylated-la-methyl steroidsof the following general formula:

wlierein lifl from the group of by drog en and hydroxyl, wherein when R is hydrogen then R is selected [mm the group consisting of hydrogen, hydroxyl, .alkoxy wherein the alkyl is a lower alkyl, and acyloxy wherein the 'a'oyYis derived from a lower aliphatic carboxyiic acid, and when R is hydroxyl then R is hydrogen, and wherein When, R is hydrogen then X is selected from the group'jc'onsisting of a single and a double bond, and whim R'is hydroxyl then X is a single bond.

Among the particularly suitable starting materials of the method of the present invention are lfia-metllyl-4- pregnene-l 7a,21'di0l 3,20 dione- (16 methyl Reichstein-S) giving over yield of IGnt methyHLpregnene- 1 1B, 17,2l -triol-3,20-dione (16a-methyl-hydrocortisone).

Among the new compounds'produced according to the method of the present'inventionare lfia-methyl-corticosterone, and 1fiwmetliyl l-lfi,l'la-oxyprogesterone.

Although the present invention is not meant to be limited as to any theory of operation, such theory is herewith given in the hopes that it will help others to better understand the invention. It appears that the 161:- methyl group makes it"difficult for the oxygenation of the ferment to take place on the backside of the D ring of the steroid molecule, or entirely prevents it, and as a result the reaction is forced to proceed in a simple manner to form the 11 ,B-hydroxylated compound.

In addition to the productiorr of llfi-hydroxyl-l6amethyl steroids the present invention also comprises the production of l4m-hydroxyl-l6a-methyl steroids. These l4m-hydroxylated la-rnelhyl steroids are obtained in lesser yield as side products in the production of 11,8- hydroxylated Mat-methyl steroids, and the new l4a-hydroxylated lfie-methyl steroids of the present invention are in and of themselves extremely valuable compounds,

3,161,662 3 4 as well as being very useful intermediate compounds in group in the l7-position, that is compounds of the folsteroid synthesis. lowing general formula:

The new 14a-hydroxylated compounds of the present invention are of the following general formula:

can:

on o 4 wherein R has the same definitionas above. Whflein R s Selected fr l g P 60115555113 Y- The method of producing the 14m-hydroxylated-l6admgen and hy Y1; 1 R is 5816565 from the methyl steroids of the present invention mainly'comprises 8 consisting of y g yd o y wherein the subjecting of acompound of the formula: the alkyl isa lower alkyl, and acyloxy wherein the acyl is derived from a lower aliphatic carboxylie acid, and

wherein when R is hydrogen then x is selected from the n,

group consisting of a single and a double bond, and when w J: R is hydroxyl then X is a single bond. r r o 5 The new compounds of the present invention include A -pregnadiene compoundswith a l'la-hydroxyl. group of the following general formula: V 4 x -CHI ' wherein R is selected from the group-consisting of hydrogen and hydroxyl, wherein R' is selected from the OH I group consisting of hydrogen and hydroxyl, and wherein when R is hydrogen then X is selected from-the group consisting of a. single and double bond and when R is hyi 4o droxyl then X is a single bond, to the action of a microwhmin R has ma same definition as abovc as we as organism selected from the group consisting of micro- Atptegnmc compounds with the l-iwhydwxyl group, of organisms of the Curvularia strain and microorganisms a of the Heliocostylum strain so as to form the correspondthe following general fommh' ing l4a-hydroxy compound, and recovering said 14a-hydroxy compound.

The resulting 14a-hydroxy compound can'be' purified in per se known manner for example by chromatography on suitable adsorption agents such as silica-gel.

In the case where R is hydroxyl and it is desired to obtain a 2l-position acylated compound, the hydroxyl is saturated, and it is desired to produce a A pregnadiene Q compound, the 1,2-position can be dehydrogenated in known manner, for example by the action of a suitable microorganism such as Bacillus lentils. whareinRhasthe same defi iti as g In order to produce the 14ahydroxy1-l6-methyl ste- The compounds of the present invention also include mids Without y -1 y y group,

A -pregnadiene compounds which do not contain a hy- Pounds of following 86116131 formula: droxyl group in the 17-position, i.e. compounds of the 50 following general formula:

50 r l a C0 43H,

(;;HI X

L I on 0 wherein R and X have the same definitions as above, the as well as M-pregnene compounds without any hydroxyl 7 starting compound should be free of any hydroxyl group in the ZI-position can be acylated in known manner. In the case where the carbon-carbon bond in the 1,2-position in the l7-position, i.e. acompound of the following general formula:

C Hi3.

wherein R and X-have the same definitions as vabove.

Similarly, if it is desired to produce a compound with a l7qn-hydroxyl group, i.e. a compound of the following general formula:

HzR

on w then the starting compound should contain the 17a-hydroxyl group, Le. a compound of the following general formula:

.CHgR.

.- steroid which -is-already msaturated-in A -position, i.e.

between-the l and 2 carbon atoms.

Reaction products whereinR is anacyloxy group, particularlythe acetoxy group, are generally not suitably pr d b s a rom th c p d n and oxygen-free 16e-inethyI sit toid 7 direct since the Zl-acylogryv grgup during. the fie entation in which the ll-oxygen or 14-ozygen function is introduced into the a molecule is saponified. It is therefore generally necessary when it is desired toproduEe-the Zl-acyIatetl final product to start with the primary hydroxylated product and then to acylate the 2l-position hydroxyl group. As a matter of fact, if the 2l-position aeylated compound is used as a starting oompoundithen the final product will probably contain a 2l-position hydroxyl group which then must be acylated if a'Zl-acyl product is desired as the final compound.

Although, as stated above, the l4a-hydroxylated-l6amethyl steroids can be produced by the action of the same microorganisms which are used to produce the 11,6-

6 hydroxylated products, that is for example by the reaction of microorganisms of the strain of the species Curvularia Iurzata, a still greater yield of the desired 14- hydroxylated product can be obtained by the use of microorganisms of the strain Hcliocostylum piriforme.

The separation of the JLB-hydroxylated product and the l4a-hydroxylated product .from each other and from the culture liquor can be achieved by fractional crystall-ization and the further purification of the hydroxy steroids precipitated in the mother liquor can be accomplished by per se known methods, for example by chromatography. A convenient method of purification is through the production of the 2l-acyl derivative. I J

The new :14a-hydroxylated-l6a-methyl steroids o fsth'e present invention are technically very valuable since :they possess a strong inflammation arresting action which even surpasses that of hydrocortisone. The particular value of the l4a.hydroxyl compounds of the present invention lies in the fact that the undesirable but typical side effect of corticoids which occurs in the treatment of inflammatory processes, such as action on the glucose addition in the liver and on the mineral metabolism, either does not occur at all with the compounds ofthe present invention or only occurs in the case of unusually high dosages. Thus, these compounds pcovide a new class of particularly valuable new acting The structural characteristic giving riseto the specific action of the new antiphlogisticswith the V hydroxyl group appears to depend on the simultaneous presence of a lfia-methyl group a l4a-hydroltyl group, since the isolated presence of a vJGm-methylgroup alone or of a Mil-hydrantgroupalqng which were e ss p o ed for c mr se PWPWi-MQQ h u h i ao h any lfi 10 h p es nt i v n i n .it was hou ht t th main structural characteristic of antiphlogistics an oxygen-function at the ll-carbon atom, which compounds however at thesamefinie had'the Iural "characte i lyri c qs s e iq Wbish i the treatment for inflammatory processes is Accordingly, the discovery that the new l4a-hydroxyl- IGa- HCfi'lYI steroids of the present invention possess these valuable properties is of great 'importanceto' n e a a T e following table fls i 'sanpafi 'q of was tion of hydrocort-isone' and t6wmethyl-4' pregnene-i4qz, 11 z 2l -triol-3,20-dione (16a-methyl-l4a-hydroxy R eichs e L Q stands for the quotient of the inflammation retardation and glycogen action.

In the case of '16nt-methyil4'-hydromy-Reichstein S the value for Q is approximatelvifll) than in the case of hydrocortisone. Thefinding clearly shows that the presence of an HIS-position hydrox'yl' grotlpis not essential for the condition of arriving at an antiphlogistic action, but that with respect to achieving only an antiphlogistic action better results can be achieved by the simultaneous presence of .a IGa-methyl group and a l4u-hydroxyl group. However, this still left unexplained the role of the l7m-hydroxyl group with respect to producing an antiphlogistic efiect a well as with respect to the suppression of the typical corticoid effect.

In further pursuit of this question compounds without a l7a-hydroxyl group were produced, i.e. compounds of the following general formula:

CHaR

wherein R has the same definition as above, that is compounds containing a 14e-hydroxyl group but not containing a l'ia-hydroxyl group, and also compounds without the A -double bond, and it was found that such compounds exhibited the same, if not stronger, anti-inflammation action as the above mentioned tested I la-hydroxlated-l6a-methyl steroids.

Thus, for example, the compound l6a-methyl-4-preg nene-14e,21-diol-3,20dione, even in the form of its 21- acetate, exhibits three times the inflammation retarding action of the free l6tz-methyl-4-pregnene-14a,17,21-triol- 3,20-dione, although the esterification of the 2l-p0sition hydroxyl according to previous experiences is supposed to weaken this particular action.

The new l7a-hydroxyl-free 16u-methyl-14a-hydroxy steroids are produced from the corresponding 14aand 17a-hydroxyl-free compounds by hydroxylation with a microorganism of the species Curvulqria Iunata or H eliocostylum piriformc in completely analogous manner to the production of the previously mentioned 16e-methyl- Ma na-dials. In addition, these compounds can be obtained by elimination of the 17a-hydroxyl group from the last mentioned diols, for example by treatment with zinc dust in dilute acetic acid.

The following examples are given to further illustrate the present invention. The scope of the invention is'not', however, meant to be examples.

Example 1 I The following procedure may be used for carrying out llp-hydroxylationz By heating for oiie half hour-at 120 C. it is sterilized and after cooling it is inoculated with a mycelium suspension of a strain of Curvularia lanata. The mycelium suspension is obtained by rinsing a 7day culture developed at 28 C. of'the fungus on 15 g. of wet maize (inoculated from a biomalt inclined agar agar culture) with 100 cc. of physiologicak saline solution.

After 2 hours of propagation at 25 C. under stirring (220 revolutions per minute) and airing (1650 liters per hour) 1.8 liters of the resulting culture's taken olf under sterile conditions and transferred into fermenter with 28.2 liters of a nutrient solution containing:

After 24 hours of cultivation 7.5 g. of the steroid to be hydroxylated is introduced into 200 cc. of ethanol tedtothespecificdetailsofthe and fermented under the same conditions. The fermentation time varies depending upon the particular steroids, as will be more apparent from the examples.

The course of the fermentation is followed by removing samples which are extracted with methylisobutyl ketone. The extract is analyzed by paper chromatography. The system used is dioxane-l-toluene/propylene glycol.

After completion of the fermentation the culture broth is filtered off under suction on a Buchner funnel and extracted three times, each time with 10 liters of methyl isobutyl ketone. The purified extracts are concentrated in a vacuum circulating evaporator and then evaporated to dryness under vacuum and under nitrogen. The residue is subjected to chromatography on silica gel*(l0% water addition).

The following table gives a summary of the results of several llp-hydroxylations carried out in the above-described manner:

TABLE 2 Fermen- Starting substance Resulting Product cation Yield,

Time, percent hours 1. lfimmethyl-t-pregnene Ith-methyl-t-pregnene- 34 30 3,2.l-dione. 1lBol-3,20-dlone. 2. tfia-methyl-i-pregnenel6a-rnethyl-4-pregncne- 32 32 17a-ol-3,20-d.iono. Elna-11101630- one. 3.16a-rnethfi-4-pregnene- 16arnethyl-4-pregnene 28 21-01%,23-dlone. 1m,2ldiol-3,20-dlone. 4. tum-math l-4- lfia-methyH-pregueue 28 81 17,2l-di0 4Lm-dione. ll8,l7a,21-tri0l\3,m-

dlone.

TABLE 3 Eluatlon Agent Solvent tor Recrys- M.P., C It... i

L chloroform: ethyl Methylene chloride! 202-314 240 14,350

acetate (3:1). isopropyl other. 2. chloroform: ethyl Methylennehlofldel 197-198 241 15.550 acetate (3:1). hexane.

146-147 240 14.300 3. chloroform: ethyl Methylene chloride Acetate:

-aoetnte (3:1). 103. 5-194 240 15.600 4. Chlomtormn.-- Ethylene chloride..- nos-20s 241 15,700

Example 2 A fermenter of stainless steel with a 50 liter capacity is charged with 30 liters of a nutrient medium containing:

. Percent Saccharose 5 Beet sugar molasses 3 1 Na'NO; 0.2

, KH PO 0.1 KCl 0.05 M so. 0.05 FeSO; 0.001 Cornsteep (pH7) 0.5

It is sterilized by heating for one half hourat 120? C. and after cooling it is inoculated with a spore suspension of Curvularia lunata, which is obtained by rinsing a 1-day maize culture (15 g. maize) with approximately cc. of physiological saline solution.

After 2 days of propagation at 25 C. under stirring (220 revolutions per minute) and airing (1.65 cubic meters per hour) 28 liters of the resulting culture are removed under sterile conditions and transferred into a 730 liter fermenter containing 470 liters of a nutrient medium containing 5% saocharose, 1% beet sugar molasses, 0.2% sodium nitrate and 0.1% of potassium dihydrogen phosphate. After 24 hours of culturing under stirring revolutions per minute) and airing (8 cubic meters per hour) g. of l6a-methyl-Reichstcin-S in 9 3.3 liters-of ethanol are added and fermented for 2-8 hours under the same conditions.

The course of the fermentation is determined by removal of samples which are extracted with methyl isobutyl ketone. The eXtracB are analyzed by paper chromatography in a system of dioxane-l-toluene/ propylene glycol.

After the end 'of the ferrnentationthe culture broth is dilteredpffunder suction on a rotating f lter and extracted .with methyl isobutyl ketone. Theextract is evaporated ,under .vacuum and 'the residue is recrystallized twice from ethyl acetate. There is thu .Qhtained l6amethyl-hy- .drocoriisone having a melting point of 210/211-213" C.

The mother liquor is evaporated and subjected to chromatography onsilica gel (10% water addition). 12 g. of the oily crystalline substance are eluated with chloroiQrm s h l assist-2& w i a e recrystallization from ethyl acetate results in 6.5 g. of pure 16a-methyl-4- a *l4e 9 3 2 -1 y n a m i point of 222/223-1224 C. a The yield corresponds to f the theoretical. Ellie ultra violetextinction amounts to 24Q=16,010.

Example 3 760 mg. of lfia-methyl-l-pregnene 14a,l7a,21 -triol 3,20-dione is permitted to stand in 6 cc. dry pyridine and 3 cc. of acetanhydride for 3 hours at room temperature and the reaction mixture is then stirred into 100 cc. of sulfuric acid at 0 C. After 1 hour the crystalline precipitated product is filtered off under suction, washed with water, dried and after recrystallization from ethyl acetate gives l6a-methyl-4-pregnene-l4a,17,2 l-triol-3,20- dione-Zl-acetate having a melting point of 215/2165- 2l8 C. and an ultra violet extinction of e =l6,500.

The yield amounting to 680 mg.=80% of the theoreti- Example 4 A 50 liter stainless steel fermenter is charged with 30 liters of a nutrient medium containing 1% yeast extract, 5% cornsteep liquor and 2% glucose (pH 7) and sterilized as described in Example 2. It is inoculated with a bacteria suspension of Bacillus Ientus, which is obtained by rinsing a bouillon agar surface of 65 cm. with 7 cc. of physiological saline solution.

After 24 hours of propagation under the conditions described in Example 2 1.8 liters of the resulting culture are removed under sterile conditions and transferred into a fermenter containing 282 liters of the same medium. At the same time the're-is added a solution of 5 g. of l6m-methy1- -pregfiene-l4a,17a2l-triol-3,20-dione in 150 cc. of ethanol and fermented under the sameconditions for 16 hours at 25' C.

The course of the fermentation is determined by removal of samples which are extracted with methyl isobutyl ketone. The extracts are analyzed polarographically. r'

The culture broth is stirred three times, each time with 10 cc. of methyl isobutyl ketone and the purified extract is evaporated in a vacuum circulating evaporator under nitrogen to dryness. The residue is recrystallized from ethyl acetate. The yield of l6m-methyl-l,4-pregnadienel4a,l7a,2i-tfl0l-3,20 di0ll6 having a melting point of 236/237-273.5 C. amountsto 3.5 g. which equals 70% of the theoretical. The ultra violet extinction is f e =l5,500

Example 5 760 mg. of lfim-methyl- 1,4 -preguadiene-l4a,l7a,2ltriol-3,20-dione are acetylated under the conditions described in Example 3 and further worked up- After recrystallization from ethyl acetate the ZI-acetate is obtained in an amount of 640 mg.=75% of the theoretical. The compound is l6a-methyl-1,4-pregnadiene-l4a,l7a,2 l-triol- '3;20-dione-2-l-acctate having a melting point of 229/ 2295-230 C. with an ultra violet extinction of Example 6 A 50 liter capacity stainless steel fermenter is charged with 30 liters of a nutrient solution containing:

Percent Saccharose 5 Beet sugar molasses l NaHCO 0.2 KH PO 0.1 KCl 4 0.5 MgSO 0.05 FeSO 0.001 .Cornsteep (pH 7') 0.5

Percent Saccharose 5 Beet sugar molasses 1 NaN0 0.2 KH PO 0.1

After 24 hours of culturing under stirring revolu tions per minute) and airing (8 cubic meters per hour) 7.5 g. of l6a-methyl-4 pregnene-2l-ol-3,20-dione in 200 cc. of ethanol are added and fermented for 28 hours under the same conditions.

The course of the fermentation is followed by removal 'of samples which are extracted with methyl isobutyl ketone. The extracts are analyzed by paper chromatog raphy in the system of dioxane-i-toluene/propylene glycol.

After the end of the fermentation the culture broth is filtered off under suction and extracted with methyl isobutyl ketone. The extract is evaporated under vacuum and the residue is subjected to chromatography on silica gel to separate the 1 l3- and l4a-hydroxylated compounds. The fraction with 16!: methyl 4-pregnene-14a,2 l-diol- 3,20-dione is acetylated as the crude product.

Example 7 methyl-4-pregnene-14a,2l-diol-3,20dione-2l-acetate having a melting point of l93l94 C.

Analysis 0:411:10; (420.5)

Calculated 7l. 7 8. 5 l9. 9 Fouu .i 70.4 9. 0 20.l14o=15,200

Without further analysis, the foregoing will so fully reveal the gist of the present invention that others can by applying current knowledge readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this invention and, therefore, such adaptations should and are intended to be comprehended within the meaning and range of equivalence of the following claims.

What is claimed as new and desired to be secured by Letters Patent is:

1. 16m-rnethyl-corticosterone. 2. A compound of the formula:

EH11! CO 3. A compound of the formula:

- wherein R is an acyloxy group the acyl of which is derived from a lower aliphatic carboxylic acid.

4. l6a-rnethyl-1,4,pregnadiene-14a,l7u,21 triol 3,20-

dione.

5. 1fia-methyl-l,4-pregnadiene-14a,17a,21 triol 3,20- dione-21-acetate.

References Cited in the file of this patent UNITED STATES PATENTS Levin et al May 17, 1955 Wettstein et al. July 22, 1958 McAleer et a1. Feb. 24, 1959 Ringold et a1. June 2, 1959 Bloom et a1. Mar. 22, 1960 OTHER REFERENces Fieser et aL: Steroids (1956), published by the Rein hold Publishing Corporation, New York, pp. 603-607 relied on. 

3. A COMPOND OF THE FORMULA: 